The cortical collecting duct of the mammalian nephron plays a critical role in maintaining Na and K balance in both physiological and pathophysiological states. The mineralocorticoid hormone aldosterone, in part, maintains this balance by regulating the rates of Na absorption and K secretion by the principal cells (the dominant cell type) of this nephron segment. Aldosterone stimulates both Na absorption and K secretion by a two-phase process: an early phase where Na entry across the luminal border is first stimulated, and is associated with cell swelling, and a late phase where the Na-K-ATPase activity (Vmax activity) of the basolateral membrane and the K conductances at both luminal and basolateral borders are up regulated. The overall goal of this proposal is to determine the mechanisms (posttranslational) of regulation underlying the actions of aldosterone on Na and K transport processes in the principal cells with particular emphasis on determining primary versus secondary actions. The specific aims are to elucidate the interrelations among ionic conductive pathways and the Na-K-ATPase, and to determine the role of Na entry, cell volume, and intracellular calcium activity in the early and late responses to aldosterone. Isolated cortical collecting ducts from rabbits will be used to assess the time-dependent actions of aldosterone in vitro on: 1) the specific Na and K conductances of the apical (luminal) and basolateral border using electrophysiological techniques, 2) the Vmax Na-K-ATPase activity of the basolateral membrane using a micro-enzymatic amplification assay, and 3) the basolateral membrane ultrastructure using quantitative stereological electron microscopy techniques. The results of the first studies will establish early versus late responses to aldosterone and provide initial evidence as to the interrelations among these parameters. In other studies, the dependency of these parameters on alterations in Na entry, cell volume, and intracellular calcium activity will be determined in the presence and absence of aldosterone to clearly define primary vs secondary responses. The results of these studies will not only provide a mechanistic model of aldosterone actions and the factors underlying the response, but also should provide new insights into the actions of other hormones where the response is associated with a change in cell volume.